1Thermo Fisher Scientific in Oslo, Norway and Austin, Texas, United States; 2Department of Immunology, Institute for Cancer Research, Oslo University Hospital, Norway; 3Centre for Cancer Biomedicine, University of Oslo, Norway; 4Department of Biosciences, Section for Physiology and Cell Biology, University of Oslo, Norway.

Exosomes are extracellular vesicular structures (50–150 nm in diameter) secreted by all cells in culture and found in all body fluids investigated to date [1]. Exosomes function in diverse biological processes, including apoptosis, antigen presentation, angiogenesis, inflammation, and coagulation [2,3]. They have been observed to activate signaling pathways, as well as to deliver nucleic acids to distant cells [4–6]. Furthermore, tumor-derived exosomes can enhance cancer progression by transferring oncogenes from tumor cells to cells lacking the oncogene [7].

Both the concentration and composition of exosomes can vary significantly during disease. The isolation and characterization of exosomes from body fluids and cell culture systems will provide important information that may be useful for early disease detection, monitoring of disease status, and the development of effective treatments for cancer, inflammation, and autoimmune diseases. Further insight into exosome biology may also accelerate the use of these nanovesicles in regenerative medicine and vaccination research and increase the efficacy of therapeutic antibodies.

Improved exosome isolation protocols under development

The established exosome isolation protocol is differential centrifugation with a final ultracentrifugation step, although density gradient or cushion [8], size exclusion [9], or precipitation [10–12] methods are also employed. Differential ultracentrifugation, however, is time consuming, requires expensive equipment, and cannot discriminate between exosome subpopulations or other particles with similar size and density such as protein aggregates, lipids, and miscellaneous nucleic acid complexes. To obtain ultrapure exosomes or to isolate potential exosome subpopulations, an immunomagnetic isolation strategy can be applied by targeting exosomal surface markers [13,14].

The aim of this study was to establish a direct method for fast, efficient, and selective isolation of exosomes from cell culture supernatants that is compatible with a wide range of downstream applications. Here we describe the use of magnetic beads coated with antibodies against the tetraspanins C9 or CD81—common exosomal markers—to isolate and characterize pre-enriched exosomes derived from SW480 (human colon adenocarcinoma) and Jurkat (human T lymphocyte) cells. Critical factors such as volume, time, and exosome concentrations were addressed in order to establish optimal and comparable isolation conditions. In addition, we have developed a method for isolating exosomes directly from cell culture medium using magnetic beads. Direct isolation methods have the potential not only to shorten the workflow, but also to reduce any artifacts or contamination that can result from enrichment procedures, producing exosomes that can be further characterized with sensitive techniques such as mass spectrometry.

Analysis of exosome markers in pre-enriched exosome samples

Typically, exosome samples are prepared using either differential ultracentrifugation or precipitation. Figure 1 shows the analysis of SW480 cell culture medium that was harvested after 24 hours, followed by centrifugation to remove debris and precipitation using the Total Exosome Isolation Reagent. As expected, the exosomes contained full-length mRNA (GAPDH and ACTB), rRNA (18S), and miRNA (Let7a and miR16), which are typical exosome cargo [15] (Figure 1A). In addition, the exosomes exhibited a size distribution comparable with that of standard samples prepared by ultracentrifugation [16] (Figure 1B), and they had the common exosome surface marker CD81 [17,18], as confirmed by anti–human CD81 antibodies and electron microscopy (Figure 1C). The exosome markers CD9 and CD81 were also detected by western blotting (Figure 1D).

Pre-enriched vesicles were then identified by flow cytometry using magnetic beads as a solid support detectable by the instrument. For equal binding kinetics, the ratio of number of magnetic beads to isolation volume was kept constant. Pre-enriched CD9- positive adenocarcinoma-derived exosomes were isolated using magnetic beads coated with anti–human CD9 antibodies followed by CD9 staining. The side/forward scatter plot demonstrates the presence of magnetic beads (Figure 2A, red data points). Isolated exosomes stained with isotypic antibody show the background autofluorescence of the beads (Figure 2B, gray peak) compared with the specific fluorescent signal exhibited by the bead-isolated exosomes stained with the R-phycoerythrin (PE) conjugate of anti–human CD9 antibody (Figure 2B, red peak). Isolation efficiency was confirmed by flow cytometric analysis of the remaining exosomes in the supernatant post-isolation. Increasing amounts of magnetic beads were used for isolation, starting with amounts equivalent to what is used for flow cytometry (1x) up to the amount of beads recommended for western blotting (25x) (Figure 2C). The flow cytometry results were confirmed by western blot analysis of exosomes prior to isolation and after isolating with anti-CD9 antibody–coated magnetic beads (Figure 2D).

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Figure 1. Analysis of precipitated exosomes obtained with the Total Exosome Isolation Reagent.(A) Precipitated exosomes released from adenocarcinoma (SW480) cells and isolated with the Total Exosome Isolation Reagent were analyzed for RNA characteristic of exosomes—mRNA (GADPH, ACTB), rRNA (18S), and miRNA (Let7a, miR16)—by qRT-PCR. Average Ct values for three independent experiments are shown (n = 3). (B) Precipitated exosomes from SW480 culture medium were analyzed by nanotracking analysis (size distribution) on the NanoSight™ LM10 instrument. The size distribution and concentration of total exosomes resuspended in PBS are shown. (C) Immunolabeling of exosomes derived from SW480 cells. Bar = 100 nm. (D) Western blot analysis of ultracentrifuged (UC) and precipitated (Prec) exosomes detected with antibody against exosomal surface protein CD9 and antibody against exosomal surface protein CD81.

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Figure 2. Flow cytometric analysis of exosomes isolated with magnetic beads targeting CD9.(A) Scatter plot of exosomes isolated with magnetic beads coated with anti–human CD9 antibody; gating is shown. (B) CD9+ exosomes isolated with magnetic beads were stained with fluorescent anti–human CD9 antibody (CD9-PE, red peak) or an isotype control (gray peak). (C) Depletion efficiency was measured by flow cytometry (signal/noise) of the supernatant after isolation for western blotting. 25x represents the amount of magnetic beads used for western blotting. (D) Exosomes were subjected to western blot analysis with anti-CD9 antibody prior to (Pre) and post (Isol) isolation with magnetic beads.

Direct isolation of exosomes from cell culture medium

Directly isolating exosomes from cell culture medium without any pre-enrichment steps would provide a fast and scalable protocol and offer several advantages for downstream applications. We investigated the use of the immunomagnetic separation method with cell culture supernatants that had not been pre-enriched for exosomes. Specifically, we used anti–human CD81 antibody–coated magnetic beads for the isolation of exosomes from T lymphocyte (Jurkat) cell culture (after an initial low-speed centrifugation step to remove cells and cell debris). Several factors were investigated. First, the impact of the volume of exosome-containing cell culture medium during isolation was addressed. Increasing the amount of exosome-containing cell culture medium (200 μL, 400 μL, 800 μL) while keeping the amount of magnetic beads constant (20 μL) provided a nonlinear (+) dose-response curve (Figure 3A). At very high volumes, the reduced concentration of magnetic beads resulted in lower binding kinetics. Second, we looked at incubation time. Increasing incubation time with a fixed amount of exosome-containing cell culture medium and magnetic beads produced a nonlinear (+) dose-response curve (Figure 3B). Lastly, we found that increasing the amount of exosomes while keeping the volume of cell culture medium and the amount of magnetic beads constant resulted in a linear (+) dose-response curve (Figure 3C).

The optimized experimental conditions were then used for the direct isolation of exosomes from cell culture medium, which were then analyzed by flow cytometry. Adenocarcinoma (SW480)–derived exosomes were isolated directly using anti-CD9 antibody–coated magnetic beads and compared with exosome samples prepared either by differential ultracentrifugation or by precipitation. To enable direct comparison of the flow analysis, the exosome input was normalized for the three ways of processing conditioned cell culture medium based on the concentration factor achieved for ultracentrifugation and precipitation, respectively. All three exosome preparation methods were then analyzed by flow cytometry (Figure 4A) and western blotting (Figure 4B) using fluorescent antibodies against CD9. Densitometry analysis showed that the exosome yields for the pre-enriched (by differential ultracentrifugation or precipitation) preparations were somewhat lower than those of the directly isolated preparations, indicating loss of exosomes during pre-enrichment steps. The isolation efficiencies of the three methods were confirmed by subjecting the supernatants remaining after initial exosome isolation to a second round of isolation (Figure 4C).

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Figure 3. Flow cytometric analysis of the effects of incubation volume, incubation time, and exosome input on exosome isolation. Exosomes derived from Jurkat cells were isolated using magnetic beads coated with anti–human CD81 antibody, followed by fluorescent anti–human CD81 antibody (CD81-PE) staining. Samples were then analyzed by flow cytometry and presented as signal/noise. (A) CD81+ exosomes were isolated from 200 μL, 400 μL, or 800 μL of cell culture medium using 20 μL of magnetic beads. (B) CD81+ exosomes were isolated from 100 μL of cell culture medium after incubation with 20 μL of magnetic beads for 5, 16, 19, or 21 hr at 4°C. (C) CD81+ exosomes were isolated from 25 μL, 50 μL, or 100 μL of added exosomes in a 100 μL total volume using 20 μL of magnetic beads.

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Figure 4. Flow cytometric analysis of CD9-positive adenocarcinoma (SW480)–derived exosomes isolated with magnetic beads directly from cell culture medium (Direct) or after pre-enrichment by ultracentrifugation (UC) or precipitation (Prec).(A) The three different exosome preparations were analyzed by flow cytometry after staining with fluorescent anti–human CD9 antibodies (CD9-PE, red peak) or an isotype control (gray peak). (B) Western blot confirmation of the results obtained by flow cytometry. (C) Exosomes were isolated for western blotting using anti-CD9 antibody–coated magnetic beads (25x the amount of beads used for flow cytometric analysis), and the remaining supernatant was subjected to a second round of exosome capture for flow cytometric analysis as described above. The red and blue bars represent the flow analysis pre- and post-exosome isolation for western blotting, respectively.

Advantages of immunomagnetic isolation protocols

The isolation and analysis of nanometer-sized exosomes is challenging. Today the general approach for exosome isolation is differential ultracentrifugation—though the protocol is not rigorously standardized— requiring several labor-intensive steps and costly equipment. Moreover, differential ultracentrifugation methods increase the risk for exosome loss and fail to discriminate well between exosomes and contaminating structures such as larger vesicles and protein/ lipid aggregates [19].

By including magnetic beads coated with antibodies specific for exosome surface proteins in the isolation protocol after pre-enrichment, we can further enhance the purity of the exosome preparation. We also demonstrate an alternative, optimized workflow for direct exosome isolation with magnetic beads that omits the pre-enrichment step altogether. Direct immunomagnetic isolation requires minimal hands-on time and produces highly purified exosome preparations with minimal loss, enabling downstream analysis and future automation opportunities.